Abstract

Salt-induced precipitation is a biological separation technique that exposes proteins to unnatural environments. Macromolecular-scale issues of activity, structure, and aggregation have been addressed as a function of governing parameters.

The effects of salt type and concentration on protein solubility and recoverable activity were studied using [alpha]-chymotrypsin ([alpha]CT) as a model protein and five salts spanning the lyotropic series. Unaccounted for salt-protein interactions and changes in protein physical properties were the likely source of discrepencies between the experimental and theoretical solubility behavior. Active protein recovery was a function of salt type, but not concentration. A salting-out performance parameter was identified; an optimum salt may exist for a particular protein.

[alpha]CT precipitates from the solubility-activity study were examined for perturbations in secondary structure via Raman spectroscopy and in active site tertiary structure via electron paramagnetic resonance spectroscopy. NaBr, KBr, and KSCN-induced precipitates had increased [beta]-sheet and decreased [alpha]-helix contents; these changes were correlated with active protein yields. Spectra of spin-labelled precipitates indicated that the active site remains intact. Molecular modelling was used to estimate changes in the dipole moment and hydrophobic surface area for the altered precipitates. A general mechanism for the precipitation of globular proteins was proposed.

The generality of secondary structure changes was explored for twelve different proteins via Raman spectroscopy. KSCN-induced precipitates exhibited increased [beta]-sheet and decreased [alpha]-helix contents; structural changes for Na[2]SO[4]-induced precipitates were less significant. The [beta]-sheet increase may occur at the expense of [alpha]-helix segments. [beta]-sheet increases were correlated with the fraction of charged residues and the surface area of the native protein. [alpha]-helix decreases were correlated with the dipole moment and helical content of the native protein.